Impact of a probiotic, inulin, or their combination on the piglets’ microbiota at different intestinal locations

2015 ◽  
Vol 6 (4) ◽  
pp. 473-483 ◽  
Author(s):  
V.A. Sattler ◽  
K. Bayer ◽  
G. Schatzmayr ◽  
A.G. Haslberger ◽  
V. Klose
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Real Time ◽  
Relative Abundance ◽  
Real Time Pcr ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Feed Additives ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

Natural feed additives are used to maintain health and to promote performance of pigs without antibiotics. Effects of a probiotic, inulin, and their combination (synbiotic), on the microbial diversity and composition at different intestinal locations were analysed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and 16S rRNA gene pyrosequencing. Bacterial diversity assessed by DGGE and/or pyrosequencing was increased by inulin in all three gut locations and by the synbiotic in the caecum and colon. In contrast, the probiotic did only affect the microbiota diversity in the ileum. Shifts in the DGGE microbiota profiles of the caecum and colon were detected for the pro- and synbiotic fed animals, whereas inulin profiles were more similar to the ones of the control. 16S rRNA gene pyrosequencing revealed that all three additives could reduce Escherichia species in each gut location, indicating a potential beneficial effect on the gut microbiota. An increase of relative abundance of Clostridiaceae in the large intestine was found in the inulin group and of Enterococcaceae in the ileum of probiotic fed pigs. Furthermore, real-time PCR results showed that the probiotic and synbiotic increased bifidobacterial numbers in the ileum, which was supported by sequencing results. The probiotic and inulin, to different extents, changed the diversity, relative abundance of phylotypes, and community profiles of the porcine microbiota. However, alterations of the bacterial community were not uniformly between gut locations, demonstrating that functionality of feed additives is site specific. Therefore, gut sampling from various locations is crucial when investigations aim to identify the composition of a healthy gut microbiota after its manipulation through feed additives.

Rapid PCR identification of Prevotella copri in an Australian cohort of pregnant women

2019 ◽  
Vol 11 (3) ◽  
pp. 228-234 ◽  
Author(s):  
Lawrence Gray ◽  
Kyoko Hasebe ◽  
Martin O’Hely ◽  
Anne-Louise Ponsonby ◽  
Peter Vuillermin ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Relative Abundance ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Birth Cohort Study ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Cost Effective Technique

AbstractGut bacteria from the genus Prevotella are found in high abundance in faeces of non-industrialised communities but low abundance in industrialised, Westernised communities. Prevotella copri is one of the principal Prevotella species within the human gut. As it has been associated with developmental health and disease states, we sought to (i) develop a real-time polymerase chain reaction (PCR) to rapidly determine P. copri abundance and (ii) investigate its abundance in a large group of Australian pregnant mothers.The Barwon Infant Study is a pre-birth cohort study (n = 1074). Faecal samples were collected from mothers at 36 weeks gestation. Primers with a probe specific to the V3 region of P. copri 16S rRNA gene were designed and optimised for real-time PCR. Universal 16S rRNA gene primers amplified pan-bacterial DNA in parallel. Relative abundance of P. copri was calculated using a 2-ΔCt method.Relative abundance of P. copri by PCR was observed in 165/605 (27.3%) women. The distribution was distinctly bimodal, defining women with substantial (n = 115/165, 69.7%) versus very low P. copri expression (n = 50/165, 30.3%). In addition, abundance of P. copri by PCR correlated with 16S rRNA gene MiSeq sequencing data (r2 = 0.67, P < 0.0001, n = 61).We have developed a rapid and cost-effective technique for identifying the relative abundance of P. copri using real-time PCR. The expression of P. copri was evident in only a quarter of the mothers, and either at substantial or very low levels. PCR detection of P. copri may facilitate assessment of this species in large, longitudinal studies across multiple populations and in various clinical settings.

scholarly journals Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

2004 ◽  
Vol 70 (8) ◽  
pp. 4971-4979 ◽  
Author(s):  
Matthias Labrenz ◽  
Ingrid Brettar ◽  
Richard Christen ◽  
Sebastien Flavier ◽  
Julia Bötel ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Baltic Sea ◽  
Real Time ◽  
Relative Abundance ◽  
Real Time Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Uncultured Bacteria

ABSTRACT We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 103 to 4.4 × 109 copies ml−1 or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 101 to 2.2 ×106 copies ml−1 or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml−1. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.

Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia

2008 ◽  
Vol 97 (10) ◽  
pp. 1376-1380 ◽  
Author(s):  
Andreas Ohlin ◽  
Anders Bäckman ◽  
Maria Björkqvist ◽  
Paula Mölling ◽  
Margaretha Jurstrand ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
The 16S Rrna Gene

scholarly journals Prevalence of Swine Hemoplasmas Revealed by Real-Time PCR Using 16S rRNA Gene Primers

2012 ◽  
Vol 74 (10) ◽  
pp. 1315-1318 ◽  
Author(s):  
Yusaku WATANABE ◽  
Masatoshi FUJIHARA ◽  
Jin SUZUKI ◽  
Fumina SASAOKA ◽  
Kazuya NAGAI ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene

scholarly journals Haemotropic Mycoplasma Infection Revealed by Real-Time PCR in Specific Pathogen-Free Rats

2014 ◽  
Vol 58 (3) ◽  
pp. 375-378 ◽  
Author(s):  
Hinako Sashida ◽  
Ryô Harasawa ◽  
Toshihiro Ichijo ◽  
Hiroshi Satoh ◽  
Kazuhisa Furuhama
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Wistar Rat ◽  
Rrna Gene ◽  
Specific Pathogen Free ◽  
Sprague Dawley ◽  
Laboratory Rats ◽  
Specific Pathogen

Abstract The presence of Mycoplasma haemomuris (haemoplasma) in blood samples collected from specific pathogen-free (SPF) laboratory rats bred in Japan was reported. Its presence was examined in Fischer 344, Sprague-Dawley (SD), and Wistar rat strains of both sexes by real-time PCR. All strains were positive for M. haemomuris infection. The 16S rRNA gene of M. haemomuris strain detected in the animals was amplified using end-point PCR. Only the entire nucleotide sequence of 16S rRNA gene of a mycoplasma strain detected in SD rats was determined and compared to those of other haemoplasmas. Our investigations suggest a wide M. haemomuris infection among the SPF rats purchased from commercial breeders in Japan.

scholarly journals Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces

2015 ◽  
Vol 81 (19) ◽  
pp. 6749-6756 ◽  
Author(s):  
Yun-Wen Yang ◽  
Mang-Kun Chen ◽  
Bing-Ya Yang ◽  
Xian-Jie Huang ◽  
Xue-Rui Zhang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Chronic Inflammatory Bowel Disease ◽  
Trinitrobenzene Sulfonic Acid ◽  
Rrna Gene ◽  
Specific Primers ◽  
Content Type

ABSTRACTMouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers forFirmicutes,Actinobacteria,Bacteroidetes,Deferribacteres, “CandidatusSaccharibacteria,”Verrucomicrobia,Tenericutes, andProteobacteriawere designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

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